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Raman Microspectroscopy Study of the Hydrolytic Degradation of Polyanhydride Network Polymers Lina Bian, Halimatu Mohammed, Devon A. Shipp, and Paul Goulet Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b04334 • Publication Date (Web): 18 Apr 2019 Downloaded from http://pubs.acs.org on April 21, 2019

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Raman Microspectroscopy Study of the Hydrolytic Degradation of Polyanhydride Network Polymers Lina Bian, Halimatu S. Mohammed, Devon A Shipp*†, and Paul J. G. Goulet‡* Department of Chemistry and Biomolecular Science, and Center for Advanced Materials Processing, Clarkson University, Potsdam, NY 13699-5810, USA Email: [email protected], [email protected] ‡ Present Address: Department of Chemistry, St. Lawrence University, Canton, NY 13617, USA

TOC Graphic Spa ally-resolved degrada on kine cs laser Raman spectrum

% Anhydride

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Cross-sec oned polyanhydride sample

g s in a e h cr t I n D ep

Time

Keywords: Polyanhydride, Raman Microspectroscopy, Hydrolytic Degradation, Biodegradable Polymer, Surface Erosion

† ORCID: Devon A. Shipp: 0000-0002-8709-1667

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Abstract Raman microspectroscopy was employed in this work to study the degradation of a polyanhydride network polymer synthesized from 4-pentenoic anhydride (PNA) and pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) monomers in order to illustrate the utility of this method and improve understanding of the polyanhydride degradation and erosion. Disk-shaped polymer samples were immersed in buffer solutions for different periods of time and hydrolytic degradation was monitored spatially and temporally via kinetic Raman studies at various depths of penetration into the samples. Erosion, meanwhile, was monitored via mass loss measurements. Dispersive Raman microspectroscopy is shown to be a particularly valuable tool for the study of the hydrolytic degradation of these materials. It confirms that these thiol-ene polyanhydrides are indeed surface eroding, while also revealing that degradation starts to occur at the core of samples on a short time scale (less than 5 h). At any given degradation time, there is a concentration gradient of unreacted anhydride, with unreacted anhydride concentration increasing from the outer edge to the center of the polymer samples. Further, the anhydride functionality is found to decrease approximately linearly with degradation time at all depths in the samples, though the degradation rate does appear to increase slightly as degradation occurs.


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Introduction Degradable polymers have significant potential for use in a wide range of applications including drug delivery vehicles,1,2 biomedical devices,3 self-cleaning surfaces,4 cell growth scaffolds,5 erodible adhesives,6 and shape memory materials.7 Consequently, the development of polymers that degrade and erode predictably in water-rich environments is currently of great interest.8,9 Our recent demonstrated that degradable networked polyanhydrides could be rapidly produced via radical-mediated thiol-ene/yne photopolymerization.10-13 These materials have been found to undergo surface erosion in aqueous environments due to hydrolytic degradation.10-12 Surface erosion is characterized by heterogeneous erosion where water diffusion into the sample is relatively slow and hydrolytic degradation occurs more quickly at the surface than in the core of the sample.14-16 Bulk erosion, on the other hand, is characterized by homogeneous erosion where degradation is slow and water diffuses quickly into the entire sample.14-16 Experimental mass loss data has revealed a long induction period that precedes the onset of erosion of these particular materials.12 A theoretical reaction-diffusion degradation model was developed by Domanskyi et al. to explain this induction period.17 The authors of the latter report noted, however, that the available macroscopic mass loss data was insufficient to gain a full understanding of the observed erosion behavior. To gain a full mechanistic understanding of the degradation and erosion of these materials, they observed that detailed microscopic chemical and structural information must be obtained. However, methods to collect such data have not been widely used in the degradable polymer field, let alone for these new polyanhydride materials. Raman scattering microspectroscopy is a powerful technique that can be utilized to partially address this need. Raman spectroscopy can be used to monitor chemical species with high temporal (less than 1s) and spatial resolution (ca. 1 μm2).18-20 It is a non-destructive 3 ACS Paragon Plus Environment

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technique that provides detailed, quantitative information about the functional groups present in chemical structures (i.e., unique “vibrational fingerprints”), and permits multiplexed detection of multiple species simultaneously.18,21 As such, there are reports of using Raman spectroscopy to assist in the structural analysis of polyanhydrides, albeit not during degradation of such polymers.22 Further, Raman spectroscopy can be used to study aqueous systems with little to no water interference, making it an ideal technique for the study of polyanhydride degradation occurring via hydrolysis in aqueous buffer solutions.22,23 Notably, infrared absorption is also a very valuable technique for the study of polymer degradation,1,24-26 but typically suffers from significant water interference and lower temporal and spatial resolution than Raman spectroscopy. In this work, we show that Raman microspectroscopy is a highly useful method for following the degradation of a polyanhydride network polymer synthesized from 4-pentenoic anhydride (PNA) and pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) monomers. The hydrolytic degradation of the polyanhydride samples in aqueous buffer solutions is monitored spatially and temporally via kinetic Raman studies at various depths of penetration into the samples (as measured from the sample edge at time = 0). These Raman measurements provide spatially resolved chemical composition data as well as kinetic data on the rates of anhydride degradation at different depths in the sample. The erosion of the polyanhydride samples is monitored in this work through mass loss data. The data presented confirm that these materials undergo surface erosion, with associated fast degradation at the sample edges. However, the data also reveal that slow degradation occurs at the core of the samples on a short time scale. The insights provided in this work are expected to contribute significantly to the development of a full mechanistic understanding of the erosion and degradation of these network polyanhydrides. 4 ACS Paragon Plus Environment

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Experimental Chemicals and Reagents. Pentaerythritol tetrakis(3-mercaptopropionate) (98%, PETMP), 4pentenoic anhydride (98%, PNA), and 1-hydroxycyclohexyl phenyl ketone (99%) were purchased from Sigma-Aldrich and used as received. Phosphate buffered saline (PBS) solutions (pH = 7.4) were prepared in deionized water with a resistivity of 18.2 MΩ·cm. Preparation of Cross-Linked Thiol-Ene Polyanhydride Samples. The photoinitiator 1hydroxycyclohexyl phenyl ketone (0.1 wt %), and the monomers PNA (1.00 mL, 5.47 mmol), and PETMP (1.34 g, 2.74 mmol) were thoroughly mixed in a vial to establish a homogeneous mixture. This mixture was then deposited into 3 disk-shaped wells (10 mm in diameter and 5 mm in height) of a polydimethylsiloxane (PDMS) mold and irradiated with UV light for 15 minutes (Oriel Instruments, 500 W mercury–xenon arc lamp, intensity ∼80 mW/cm2 measured by a Hioki 3664 optical power meter). Mass Loss Erosion Studies. Cross-linked disk-shaped polyanhydride samples (10 mm diameter, 5 mm height) were degraded in 100 mL PBS buffer solutions (pH=7.4) at 37 °C. The buffer solution was changed every 4 hours to maintain a constant pH. The samples were carefully removed from the buffer solution using tweezers and surface water was wiped clear using a nonlint tissue. The mass of the samples was measured after 0, 4, 8, 10, 12, 16, and 24 h of degradation in buffer, and the values reported are the mean values of three replicate samples. The % mass remaining was calculated with respect to the initial mass of each sample. Samples that were allowed to degrade for 48 hrs no longer had the disk shape and could be dissolved in chloroform, thus indicating no crosslinking remained. 5 ACS Paragon Plus Environment

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Raman Scattering Studies of Degradation. Cross-linked, disk-shaped polyanhydride samples were degraded in 100 mL PBS buffer solutions (pH=7.4) at 37 °C. The buffer solution was changed every 4 hours to maintain the pH, and Raman analysis was performed on the samples after 0, 5, 10, 16, and 24 hours of degradation. Upon removal from the buffer, remaining droplets of solution were gently wicked away from the samples and the disks were carefully crosssectioned (2 cuts generating a flat rectangular slab) using a clean, sharp blade. Raman scattering spectra were then collected from the cross-sections at various depths of penetration into the sample, as measured from the original sample edge (0, 100, 200, 300, 400, 500, 600, 800, 1000, 1500, 2000, and 2500 µm). For each depth, spectra were recorded at three different spots. All values for the percentage of anhydride remaining were determined using normalized, integrated areas under the asymmetric C=O stretching peak of the anhydride group (1812 cm-1). The integrated anhydride peak intensity was normalized relative to the integrated intensity of the C-H stretching peaks (2800 – 3100 cm-1), and the percentages of anhydride remaining were calculated relative to time 0 h. All Raman scattering spectra were collected using a Renishaw inVia Raman spectrometer with 514 nm laser excitation. The laser was focused to the sample using either a 5x or a 20x objective, and an incident power of ca. 5 mW. The spectrometer was calibrated before each use using a clean silicon wafer. Results and Discussion Using the approach we have previously reported, polyanhydride network polymers were generated in this work from PNA and PETMP monomers via radical-mediated thiol-ene photopolymerization (Scheme 1).12 This polymerization takes advantage of highly efficient thiolene chemistry, and leads to essentially 100% monomer reaction in several minutes (See Figures SI-1 and SI-2 in Supporting Information for Raman spectra taken during polymerization and 6 ACS Paragon Plus Environment

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monomer conversion plots). It proceeds via a step-growth mechanism, generating a highly uniform network structure.10-12 The crosslinked polyanhydrides generated from these monomers have been shown to erode in water rich environments as a result of hydrolytic degradation.10-12 In this process, anhydride groups in the polymer are hydrolyzed, generating a water soluble small molecule degradation product with carboxylic acid functional groups.10

Scheme 1. Thiol-ene polymerization generating crosslinked polyanhydride from 4-pentenoic anhydride (PNA) and pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) monomers, and subsequent hydrolytic degradation of crosslinked polyanhydride into small molecule degradation product.

The percent mass of crosslinked polyanhydride remaining as a function of degradation time for disk-shaped polyanhydride samples was analyzed in order to correlate the mass loss, 7 ACS Paragon Plus Environment

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degradation and loss of the anhydride functionality over time, the latter parameter measured by Raman spectroscopy (vide infra). The mass loss data are shown in Figure 1 (three samples were measured and averaged for each time point), and replicate our previously published data extremely well.12 For the initial 5 hours of degradation, the polymer samples gained approximately 1.5% weight compared with the starting mass. This minor mass increase is due to uptake of water in the polymer network. Between 8 and 12 hours, a slight decrease of mass was observed, as erosion started to occur. Beginning at 12 hours, the mass remaining linearly decreased by ca. 5% per hour for these particular disk-shaped samples. These observations agree qualitatively and quantitatively with earlier reports that studied the erosion behavior of these materials.10,12 Furthermore, the mass loss data can be correlated with images of the surface at 0, 10 and 15 hours which were collected using scanning electron microscopy (SEM; see Figure SI3 of Supporting Information). These images show that the initially smooth surface becomes rough at 8 hours, when the mass has increased slightly presumably due to some uptake in water, and then becomes smooth again once the mass loss rate is linear. Hence, it is clear that the degradation behavior of these polyanhydrides is not a simple surface erosion process. Therefore, measurement of the anhydride content by Raman spectroscopy in conjunction with the macroscopic information from mass loss and SEM data is expected to yield a better understanding of the degradation and erosion processes.


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Figure 1. Plot of the percent mass of crosslinked polyanhydride remaining as a function of degradation time for disk-shaped polyanhydride samples degraded in PBS buffer solution (pH = 7.4) at 37 °C. Data points (hrs, %) are averages of 6 replicates: (0.100, 0); 4, 100.8); (8, 100.8); (12.5, 99.5); (16, 78.2); (21, 60.4); (25, 49.9); (29, 30.0); (33, 20.4), (37, 10.9).

Raman scattering spectra of the crosslinked polyanhydride, degradation product (i.e., non-solublized material left after 48 hrs after degradation), and monomers are displayed in Figure 2; from this data several identifying features can be observed that will allow such spectra to be used to follow the anhydride degradation process in these polymers. Peak identifications were initially done using the monomer species. For PNA (Figure 2 c), the sharp peak at 1641 cm1

is due to the C=C stretching vibrations of vinyl groups, while the peaks at 1750 cm-1 and 1815

cm-1 are due to C=O stretching vibrations of anhydride groups. For PETMP (Figure 2 d), the peak at 1733 cm-1 is assigned to C=O stretching vibrations of ester groups, and the strong peak at 2568 cm-1 is due to S-H stretching vibrations of thiol groups. After thiol-ene polymerization, as


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shown in the spectrum of the crosslinked polyanhydride (Figure 2 a), the S-H and C=C peaks of the monomers are no longer present. The 1738 cm-1 peak can be assigned to the overlap of the ester peak (1733 cm-1) and the second anhydride peak (1750 cm-1). The anhydride peak is slightly shifted from 1815 cm-1 to 1812 cm-1. This peak is not observed after degradation (Figure 2 b), due to the complete hydrolysis of polyanhydride, but there is a broad peak at ~1700 cm-1 that is assigned to the C=O stretch of the carboxylic acid product from hydrolysis. Raman spectra also confirm that the samples have a uniform composition throughout the sample since spectra taken at the top surface, interior, and bottom surface of the disks are identical (see Figure SI-2), regardless of the initial monomer composition used in the polymerization. In fact, Raman spectra are sensitive enough to readily observe S-H or C=C peaks when a slight excess of thiol or ene monomers, respectively, are used in the polymerization (Figure SI-2). Thus, Raman microspectroscopy can be utilized to analyze the polymerization and degradation processes of this crosslinked polyanhydride, specifically that the anhydride C=O peak at 1812 cm-1 can be used to determine anhydride content.


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C=O (ester)

C=O (acid)

C=O (anhydride)

C=O (ester)

C=C C=O (anhydride)

S-H

C=O (ester)

Figure 2. Raman scattering spectra of (a) crosslinked polyanhydride [1738 cm-1 peak due to overlap of the ester peak (1733 cm-1) and the second anhydride peak (1750 cm-1); 1812 cm-1 anhydride C=O], (b) fully degraded polyanhydride sample (sample after 48 hrs), (c) 4-pentenoic anhydride (PNA) [1641 cm-1 C=C stretch; 1750 cm-1 and 1815 cm-1 anhydride C=O stretch], and (d) pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) [1733 cm-1 ester C=O stretch 2568 cm-1 S-H stretch].

Raman scattering spectra of the surface of a polyanhydride sample degraded in PBS buffer are shown in Figure 3 and clearly show the hydrolysis of the anhydride moieties since the anhydride peak at 1812 cm-1 decreases, and a broad carboxylic acid vibration around 1700 cm-1 gradually increases, overlapping with the peak 1738 cm-1. Because erosion (i.e., mass loss) starts after 10 hours, the first three Raman scattering spectra (Figure 3 a-c) were collected from the original surface of the sample. Each of these spectra clearly show the 1812 cm-1 anhydride peak, although it diminishes in size over time, and there is noticeable broadening of the peak at ~1700 cm-1 due to carboxylic acid production. However, the spectrum (Figure 3 d) recorded from the 11 ACS Paragon Plus Environment

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sample at 16 hours is from a fresh surface due to erosion. At this time point, the 1812 cm-1 peak is barely discernable. After 48 hours, the fully degraded product, which while not completely dissolved in the PBS buffer was soluble in chloroform and thus not crosslinked, was collected and its Raman spectrum measured (Figure 3 e). This spectrum clearly indicates there are no anhydride groups left.

Figure 3. Raman scattering spectra of the surface of a polyanhydride sample degraded in PBS buffer solution for (a) 0, (b) 5, (c) 10, (d) 16, and (e) > 48 h (fully degraded).

The polyanhydride degradation process was further monitored using Raman microspectroscopy with high spatial and temporal resolution. The selected hydrolysis time samples (0, 5, 10, 16, and 24 h) were sectioned into slabs (Figure 4 a) for this study. The white


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light montage image (Figure 4 b) taken from the Raman microscope shows a rectangle (ca. 10000 μm  5000 μm) surface of the cross-section of the non-degraded polyanhydride sample. The dotted lines in the image from the top to the center of the sample represent the approximate lines along which the Raman spectra were collected. Spectra were recorded at various depths of penetration into the sample, as measured from the sample edge (0, 100, 200, 300, 400, 500, 600, 800, 1000, 1500, 2000, and 2500 µm). For each depth, spectra were recorded at three different spots.

Figure 4. (a) Illustration of the cross-sectioning of disk-shaped polyanhydride samples for Raman microspectroscopy, and (b) white light montage image collected using Raman microscope showing a representative polyanhydride sample cross-section. The dotted lines in (a) and (b) signify approximately the lines along which Raman spectra were recorded at various times.

Using this approach we were able to monitor the % anhydride as a function of depth of penetration into the samples (Figure 5). This spatial degradation data show that degradation rates depend on the depth. The average percentage of anhydride in a newly prepared sample (time 0 hour) was designated to be 100%. However, there is only ca. 96% anhydride for the outer edge 13 ACS Paragon Plus Environment

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of this sample, likely due to hydrolysis caused by atmospheric water. After a sample was immersed in the PBS buffer for 5 hours at 37 °C, there was about 81% anhydride remaining on the outer edge, gradually increasing toward the interior of the sample to 92% in the center. For the sample at 10 hours, the percentage of remaining anhydride at the outer edge to the center of the sample is 54% to 86%. These results demonstrate that the degradation at the surface is faster than in the interior of the samples, which is typical for surface-eroding materials. After 16 hours, the original surface of the sample (depth 0 µm) had been eroded. The percentage of remaining anhydride at the new surface (depth 200 µm) to the center was determined to range from 31% to 77%. For the 24 hours degradation sample, which was much smaller than the original sample (60% by weight), the percentage of remaining anhydride at the new surface (depth 800 µm) to the center ranged from 32% to 71%.

Figure 5. Plots of % anhydride as a function of depth of penetration into the samples (as measured from the sample edge at time = 0) at 0 (blue), 5 (red), 10 (green), 16 (purple), and 24 h (orange). 14 ACS Paragon Plus Environment

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In Figure 6, the % anhydride is plotted as a function of time for different depths, thus providing a more standard kinetic picture for each depth monitored compared to the data plotted in Figure 5. We note that the relatively large error bars are due to the rough surface after sectioning and the hydrolysis of samples. This plot shows that the percentage of anhydride at different depths of penetration into the samples decreases in an approximately linear fashion, though the degradation rate does appear to increase slightly as degradation occurs. These data clearly show that the degradation rate decreases with increasing depth into the sample. We suggest that the degradation is essentially a heterogeneous reaction where there is a zero-order dependence on the concentration of the two reactants (the anhydride and water), and the rate of reaction depends on the surface reaction sites available, i.e., reactive surface area. As such, the degradation rate depends on the degree of degradation. Near the surface (small values of d in Figure 6) the degradation rates are larger (e.g., d = 200 m, rate ~ 1 × 10-3 % s-1), due to higher degrees of degradation and the associated larger reactive surface areas and greater water penetration. Deeper into the sample the rates are lower (e.g., d = 2500 m, rate ~ 4 × 10-4 % s-1), due to lower degrees of degradation and the associated smaller reactive surface areas and lower water penetration. While such rate differences are the basis of model surface-erosions, the fact that the degradation rates in the interior of the sample are non-zero means that these materials are not undergoing idealized surface erosion.


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Figure 6. Plots of % anhydride as a function of time at depths of penetration into the samples (as measured from the sample edge at time = 0) of 0 (black), 200 (pink), 400 (purple), 600 (light blue), 800 (dark blue), 1000 (green), 1500 (yellow), 2000 (orange), and 2500 μm (red).

Since the Raman microspectroscopy data indicates that slow degradation of the anhydride occurs at the core of the sample even at short times, which is somewhat unexpected since a purely surface-eroding material should not undergo degradation deep into the sample, especially at short times, it is likely that some water is penetrating deep into the sample (i.e., to the center). For example, it is clear that in the present case that the % anhydride at the center of sample is reduced within the first 5 hours (by ~8%) and 10 hours (by ~13%), even though the overall dimensions appear to not change over this timeframe. We noticed in our previously published modeling17 that even a small amount of water may lead to some degradation well before 16 ACS Paragon Plus Environment

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significant mass loss occurs (i.e., between 5-10 hours). We therefore set out to find evidence of water in the center of the sample. Infrared spectroscopy is better suited to do this than Raman spectroscopy, so we used attenuated total reflectance infrared (ATR-IR) spectroscopy on slices of polyanhydride that were sectioned from the center of a sample that had been kept in aqueous buffer solution for 5 hours. The sample was then thoroughly dried by placing in vacuo for 24 hours, and the IR spectrum was remeasured. These spectra are shown in Figure 7 (note that ATR-IR spectra of the as-made polymer, water, the undried and dried polymer, and a fullydegraded sample can be found in the Supporting Information, Figure SI-4). The spectrum of the undried sample shows a broad signal between ~2500-3600 cm-1, which is indicative of the presence of water or carboxylic OH groups. After drying, however, this broad peak is significantly reduced; confirming that water does indeed penetrate to the center of the samples on a short timescale. 100

90

Transmittance (%)

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70

60

50 4000

PAH after 5 hours - not dried PAH after 5 hours - dried

3600

3200

2800

2400

2000

1600

-1

1200

800

Wavenumbers (cm )

Figure 7. ATR-IR spectra of polyanhydride before and after drying.


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These results, coupled with the non-linear erosion profiles previously observed (e.g., Figure 1), show that the erosion and degradation processes of these polyanhydrides are complicated by the deep penetration of water, even when little or no mass loss is observed. Such complexity is not entirely unexpected; as noted by other researchers, the dominant erosion mechanism that occurs at any given time is dependent on a number factors and polymer properties,15 such as crosslinking, solubility and crystallinity. Modeling of polymer erosion has often been categorized into either surface-erosion or bulk-erosion (or a transition from surface to bulk erosion). It appears that erosion of this material does not fall into either of the pure categories, but rather is an intermediate case that more closely resembles surface erosion. This study shows that Raman microspectroscopy is particularly useful in examining the degradation chemistry of these polyanhydrides, and that these polymers undergo heterogeneous degradation because hydrolysis is faster at the outer edge than in the middle, thus confirming the general surface erosion mechanism of this type of polyanhydride. We anticipate that the Raman microspectroscopy data will enhance our modeling approaches to fully understanding the degradation and erosion behavior of these and other degradable polymers.

Conclusions The thiol-ene polyanhydrides studied here have potential medical, biomedical and pharmaceutical applications due to their interesting degradation and erosion properties. The Raman spectroscopy study presented here confirms that the thiol-ene polyanhydride degradation is indeed undergoing surface erosion, but shows that degradation also occurs at the core of samples at short times, well before measureable mass loss occurs. Additionally, Raman


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microspectroscopy was able to determine that the percentage of anhydride remaining decreased with degradation time at all depths in the sample, although rates of degradation were faster at the outer edges of the samples compared to the center of the samples. The insights and data reported here are expected to contribute significantly to the development of a full mechanistic understanding of the degradation and erosion of these new polyanhydride materials, which will follow in a forthcoming publication. Acknowledgments We thank the Department of Chemistry and Biomolecular Science and the School of Arts and Sciences at Clarkson University for their support. We also acknowledge the Center for Advanced Materials Processing (CAMP), a New York State Center for Advanced Technology, at Clarkson University, for continued support and access to instrumentation. We thank Dr. Katie Poetz for assistance with the SEM measurements. Finally, we thank Professor Vladimir Privman and Sergii Domanskyi for their assistance and helpful discussions.


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Galperin, A.; Long, T. J.; Ratner, B. D. Degradable, Thermo-Sensitive Poly(N-isopropyl acrylamide)-Based Scaffolds with Controlled Porosity for Tissue Engineering Applications. Biomacromolecules 2010, 11, 2583-2592.

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Lawton, M. I.; Tillman, K. R.; Mohammed, H. S.; Kuang, W.; Shipp, D. A.; Mather, P. T. Anhydride-Based Reconfigurable Shape Memory Elastomers. ACS Macro. Lett. 2016, 5, 203-207.

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Poetz, K. L.; Shipp, D. A. Polyanhydrides: Synthesis, Properties and Applications. Aust. J. Chem. 2016, 69, 1223-1239.

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Basu, A.; Domb, A. J. Recent Advances in Polyanhydride Based Biomaterials. Adv. Mater. 2018, 30, 1706815.


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(10) Shipp, D. A.; McQuinn, C. W.; Rutherglen, B. G.; McBath, R. A. Elastomeric and degradable

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photopolymerization. Chem. Commun. 2009, 6415-6417. (11) Rutherglen, B. G.; McBath, R. A.; Huang, Y. L.; Shipp, D. A. Polyanhydride Networks from Thiol−Ene Polymerizations. Macromolecules 2010, 43, 10297-10303. (12) Poetz, K. L.; Mohammed, H. S.; Snyder, B. L.; Liddil, G.; Samways, D. S. K.; Shipp, D. A. Photopolymerized Cross-Linked Thiol-Ene Polyanhydrides: Erosion, Release, and Toxicity Studies. Biomacromolecules 2014, 15, 2573-2582. (13) Durham, O. Z.; Poetz, K. L.; Shipp, D. A. Polyanhydride Nanoparticles: Thiol-Ene 'Click' Polymerizations Provide Functionalized and Cross-linkable Nanoparticles with Tuneable Degradation Times. Aust. J. Chem. 2017, 70, 735-742. (14) Gopferich, A. Mechanisms of polymer degradation and erosion. Biomaterials 1996, 17, 103-114. (15) Gopferich, A.; Tessmar, J. Polyanhydride degradation and erosion. Adv. Drug Deliver. Rev. 2002, 54, 911-931. (16) Lyu, S.; Sparer, R.; Untereker, D. Analytical solutions to mathematical models of the surface and bulk erosion of solid polymers. J. Polym. Sci. Part B: Polym. Phys. 2005, 43, 383-397. (17) Domanskyi, S.; Poetz, K. L.; Shipp, D. A.; Privman, V. Reaction-diffusion degradation model for delayed erosion of cross-linked polyanhydride biomaterials. Phys. Chem. Chem. Phys. 2015, 17, 13215-13222. (18) Handbook of Vibrational Spectroscopy; Chalmers, J. M.; Griffiths, P. R., Eds.; John Wiley & Sons, Ltd.: Chichester, UK, 2002.


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(19) Goulet, P. J. G.; Aroca, R. F. Distinguishing Individual Vibrational Fingerprints: SingleMolecule Surface-Enhanced Resonance Raman Scattering from One-to-One Binary Mixtures in Langmuir−Blodgett Monolayers. Anal. Chem. 2007, 79, 2728-2734. (20) Goulet, P. J. G.; Pieczonka, N. P. W.; Aroca, R. F. Mapping single-molecule SERRS from Langmuir-Blodgett monolayers on nanostructured silver island films. J. Raman Spectrosc. 2005, 36, 574-580. (21) Long, D. A. The Raman Effect: A Unified Treatment of the Theory of Raman Scattering by Molecules; John Wiley & Sons, Ltd.: Chichester, UK, 2002. (22) Tudor, A. M.; Melia, C. D.; Davies, M. C.; Hendra, P. J.; Church, S.; Domb, A. J.; Langer, R. The application of Fourier transform Raman spectroscopy to the analysis of poly(anhydride) homo- and co-polymers. Spectrochimica Acta 1991, 47A, 1335-1343. (23) Herrera, M. G.; Padilla, C. A.; Hernandez-Rivera, P. S. Surface Enhanced Raman Scattering (SERS) Studies of Gold and Silver Nanoparticles Prepared by Laser Ablation. Nanomaterials 2013, 3, 158-172. (24) Davies, M. C.; Khan, M. A.; Domb, A.; Langer, R.; Watts, J. F.; Paul, A. J. The Analysis of the Surface Chemical-Structure of Biomedical Aliphatic Polyanhydrides Using XPS and TOF-SIMS. J. Appl. Polym. Sci. 1991, 42, 1597-1605. (25) Krishnan,

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Scheme 1. Thiol-ene polymerization generating crosslinked polyanhydride from 4-pentenoic anhydride (PNA) and pentaerythritol tetrakis(3-mercaptopropionate) (PETMP) monomers, and subsequent hydrolytic degradation of crosslinked polyanhydride into small molecule degradation product. 165x149mm (300 x 300 DPI)

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Figure 1. Plot of the percent mass of crosslinked polyanhydride remaining as a function of degradation time for disk-shaped polyanhydride samples degraded in PBS buffer solution (pH = 7.4) at 37 °C. Data points (hrs, %) are averages of 6 replicates: (0.100, 0); 4, 100.8); (8, 100.8); (12.5, 99.5); (16, 78.2); (21, 60.4); (25, 49.9); (29, 30.0); (33, 20.4), (37, 10.9). 111x122mm (300 x 300 DPI)


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Figure 2. Raman scattering spectra of (a) crosslinked polyanhydride [1738 cm-1 peak due to overlap of the ester peak (1733 cm-1) and the second anhydride peak (1750 cm-1); 1812 cm-1 anhydride C=O], (b) fully degraded polyanhydride sample (sample after 48 hrs), (c) 4-pentenoic anhydride (PNA) [1641 cm-1 C=C stretch; 1750 cm-1 and 1815 cm-1 anhydride C=O stretch], and (d) pentaerythritol tetrakis(3mercaptopropionate) (PETMP) [1733 cm-1 ester C=O stretch 2568 cm-1 S-H stretch]. 119x145mm (300 x 300 DPI)


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Figure 3. Raman scattering spectra of the surface of a polyanhydride sample degraded in PBS buffer solution for (a) 0, (b) 5, (c) 10, (d) 16, and (e) > 48 h (fully degraded). 110x148mm (300 x 300 DPI)


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Figure 4. (a) Illustration of the cross-sectioning of disk-shaped polyanhydride samples for Raman microspectroscopy, and (b) white light montage image collected using Raman microscope showing a representative polyanhydride sample cross-section. The dotted lines in (a) and (b) signify approximately the lines along which Raman spectra were recorded at various times. 133x122mm (300 x 300 DPI)


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Figure 5. Plots of % anhydride as a function of depth of penetration into the samples (as measured from the sample edge at time = 0) at 0 (blue), 5 (red), 10 (green), 16 (purple), and 24 h (orange). 116x143mm (300 x 300 DPI)


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Figure 6. Plots of % anhydride as a function of time at depths of penetration into the samples (as measured from the sample edge at time = 0) of 0 (black), 200 (pink), 400 (purple), 600 (light blue), 800 (dark blue), 1000 (green), 1500 (yellow), 2000 (orange), and 2500 μm (red). 104x140mm (300 x 300 DPI)


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Figure 7. ATR-IR spectra of polyanhydride before and after drying. 165x113mm (300 x 300 DPI)


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Table of Contents Graphic 128x63mm (300 x 300 DPI)


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